EVALUATING MORPHOLOGICAL DIFFERENCES BETWEEN TWO MICROBIAL MAT

MORPHOTYPES FROM LITTLE AMBERGRIS CAY AS A TOOL FOR STUDYING THE

GEOLOGICAL RECORD

by

Sydney Riemer

Undergraduate Thesis

Abstract

Fossilized microbial mats are important records of early life on Earth. The changing

macroscale morphology of these lithified microbial mats through time has been interpreted as

either the result of biological evolution or changing environmental conditions. Microbial mats

from active carbonate platforms serve as modern analogues and possible precursors to lithified

microbial mats, thereby allowing us to test hypotheses about microbial mat formation, growth,

and morphology. In this study, we apply geological methods used to study the sedimentary

record of ancient microbial mats to two modern microbial mat morphotypes–the polygonal and

document differences in microfaunal abundances and filament widths between the surface layers

and old counterparts of the polygonal and flat mats, indicating environmental changes through

time. These results imply that mat morphologies are influenced by environmental conditions

rather than microbial communities, and are consistent with findings from gene sequencing

studies. Overall, the results of this study demonstrate that distinct mat morphologies occur not

because of biological diversity, but environmental factors and have implications for studying

paleoenvironmental change in sedimentary records of lithified microbial mats.

1. Introduction

1.1 Microbial Mats and Stromatolites

yet can be difficult to interpret due to the post-depositional alteration of rocks and sediments by

tectonic activity and diagenesis, which distort paleoenvironmental records. Therefore, when

studying the geological record, it is helpful to look at modern systems that may serve as

analogues to the geologic feature being studied. One such geologic feature that has benefited

as sediment is accreted (Grotzinger and Knoll, 1999), although they can also grow by abiotic

processes (Grotzinger and Rothman, 1996). The two commonly accepted mechanisms for

microbially-mediated stromatolite growth and accretion are: (1) the trapping and binding of

sediments in microbial mats and (2) the precipitation of carbonates and other inorganic materials

through the activity of microbial mats (Grotzinger and Knoll, 1999). Stromatolites are significant

because they can be archives of ancient organisms, ecosystems, and environments on the early

Earth and throughout geologic time. However, both biological and environmental processes

influence stromatolite forms found in the geological record. Yet, diagenetic alteration of rocks

makes it difficult to identify the taxonomic and metabolic diversity of microorganisms found in

stromatolites (Knoll et al., 2013). This leads to the question of whether variations in stromatolite

life with diverse lifestyles and metabolisms. Microbial mats also contain the metabolic

byproducts of these microorganisms as well as trapped sediments, mineral precipitates, and

allochthonous material (Des Marais, 2003). They are found all over the Earth in environments

ranging from Antarctic lakes to the deep sea. Microbial mats typically adhere to a pattern of

vertical stratification. The two layers found in most mats exposed to light are a green layer and a

pink layer. The top green layer is typically on the mm- to cm- scale and is host to

photoautotrophs and heterotrophs. Of these, the most important for mat-building are

photosynthetic cyanobacteria (Knoll et al., 2013). In the pink layer below the green layer, light

penetrates but oxygen does not because it is consumed in the top green layer. Therefore, this

enter the geologic record. This vertical stratification pattern is important to consider because

modern microbial mats serve as analogs to those preserved in the geologic record, and therefore

we can compare what diversity we see in the surface layers of modern microbial mats to the

lower layers that contain previous generations of the mats to learn what aspects of biodiversity

are preserved in lithified microbial mats. Thus, microbial mats in carbonate environments offer

insights into the physical, chemical, and biological processes that occur as these diverse

ecosystems go through various stages of degradation and decay during burial, and how these

various processes affect mat structure and stromatolite formation.

relief of the lamina, and therefore it is the microstructural characteristics of stromatolites that are

studied to determine how different stromatolite morphologies originate. Microstructural

characteristics that are often studied include cyanobacterial filament orientation, size and shape,

and inorganic-precipitate fabric (Knoll et al., 2013; Grotzinger and Knoll, 1999). Though

stromatolites have been studied for over a century, it is still unclear what processes are most

important in influencing microbial mat and stromatolite morphology. Elucidating this can help

researchers determine what components of stromatolite morphology represent information about

biological diversity and evolution, global and local environmental change, or both.

Researchers have come at the stromatolite morphogenesis problem from a variety of angles using

different methods and techniques. Grotzinger and Knoll (1999) use numerical simulations to

Formation in Canada, which records intertidal to supratidal carbonate deposition (Knoll et al.,

2013). They found stromatolites of varying morphologies there, and by analyzing which

microfabrics are correlated with different microfossil assemblages, they determined that

stromatolite microfabrics and therefore macro-morphology vary depending on whether the mat is

composed of mostly coccoid bacteria, vertically oriented cyanobacteria filaments, or both

horizontally and vertically oriented filaments. As a result, their conclusion was that

microstructural fabrics of stromatolites, and therefore also morphology, are influenced by the

microbial populations. Filament orientation is also one of the characteristics most influential to

the structure. Additionally, they showed that stromatolites with the most diverse microfossil

populations occur in depositional environments where they are more frequently submerged, and

more accurate estimate of the identity and diversity of microbial populations in microbial mats.

Because many microorganisms, especially cyanobacteria, have morphological features that may

seem to distinguish species, but which are homoplastic, gene sequencing is crucial for the

taxonomic classification of microbial mat organisms (Trembath-Reichert et al., 2016). A recent

study from Little Ambergris Cay, Turks and Caicos Islands, the same field site from this study,

used 16S rRNA gene sequencing and compared the cyanobacterial and non-cyanobacterial

populations of two microbial mat morphotypes to test the hypothesis of whether microbial

in the non-cyanobacterial populations of the two mat types, and that the mat type found in the

interior of the lagoon where there is less hydrodynamic variability (polygonal mat) is more

diverse than the mat type found closer to the main tidal channel that has more hydrodynamic

activity and disturbance happening around it (flat mat). Lastly, Trembath-Reichert et al. (2016)

hypothesized that the polygonal mat morphology might develop from an initially flat mat

morphology, where diversity increases over time when there is little environmental disturbance

to the mat. This hypothesis was based on previous work from Wanless et al. (1988) that

categorized the mat morphotypes based on the presence of cyanobacteria with different

colonization strategies. The flat mat had a “Schizothrix” morphology that was described as a

rapid colonizer whereas the polygonal mat had a “Scytonema” morphology that was slower

1.3. Objectives and Hypotheses

The purpose of this study is to use modern microbial mats with distinct morphologies to

evaluate differences between the microbial populations of the two mats types, and what aspects

of these differences might enter the geological record and yield information about either

biological evolution or environmental change. In order to do so, I investigate microfaunal

assemblages in two microbial mat morphotypes from Little Ambergris Cay, Turks and Caicos

Islands, British Overseas Territories to test hypotheses about biological and environmental

influences on microbial mat morphology, as well as the potential preservation of these mat

communities in the geological record. Little Ambergris Cay is a carbonate platform where

Little Ambergris Cay mats particularly useful for testing hypotheses on the processes of

degradation and decay that influence what stromatolites in Precambrian carbonates record.

variety of morphotypes. The three observed morphotypes are classified based on texture and

morphology, and vary with elevation (Stein et al., 2016). The mat type occurring at the lowest

elevation is the permanently submerged extracellular polymeric substance (EPS) coated flat mat.

The polygonal mats with tufts of cyanobacterial sheaths are tidally submerged, and the blister

mats are only submerged during storms (Fig. 1; Stein et al., 2016). This study considers the EPS-

and imaged, because while it is important to look at the upper, live mat layers, the lower old mat

could also be representative of what might enter the geological record. By comparing the old

layers to the upper layers of the active mat, we can see what information is lost upon decay and

degradation of the mat and what preservational biases there may be. Imaging software

(JMicroVision 1.2.7) was used to do point-counts of mat features and organisms and make

measurements of bacteria filament widths. Though it was shown using 16S rRNA gene

sequencing that the polygonal mats are taxonomically more diverse than the flat mats,

(Trembath-Reichert et al., 2016), researchers studying lithified microbial mats in the rock record

do not have the ability to use gene sequencing to determine which microbial species are present

and how diverse the population is. Instead, many studies of lithified microbial mats and

characteristics, while obtaining a result that may be more useful and have more application for

geological studies of ancient microbial mats. Statistical analyses were performed in Python on

the point-count and filament width data to test hypotheses about how mat macrostructure

provides information about microbial communities and/or environmental conditions and how this

information might be recorded in the geological record.

and filament width data of probable cyanobacteria. First, based on the morphological diversity of

organisms, we determine what morphological differences there are overall between the two mats.

Next, we determine if the cyanobacteria population of the polygonal mat is indistinguishable

from that of the flat mat based on the widths of the cyanobacteria filaments. Finally, we

2. Methods

2.1 Study site

Little Ambergris Cay is an uninhabited island on the Caicos Platform with an area of

about 6 km x 1.6 km (Fig. 2). It is at an elevation of 1-1.5 m above sea level and is framed by a

bedrock rim of oolitic grainstone (Gomes et al., in revision; Orzechowski et al., 2016; Stein et

al., 2016). The interior basin of the island is dominated by microbial mats and mangroves (Stein

et al., 2016). Water depth varies tidally by up to 50 cm, although this variability is less in the

by a 1 mm lighter green-white layer that contains calcium carbonate grains, a 2 mm green layer

with filaments, and lastly, a 2-4 mm pink layer. Beneath these layers are alternating layers of

brown, filamentous layers and ooid layers (Fig. 4). The flat mat, sample CC3, was collected on

July 4, 2016 (Fig. 3 and 5). The top pigmented layers of CC3 are a 2-3 mm thick, grainy lime

green layer followed by a 1 mm pink layer (Fig. 5). The unpigmented layers are brown,

filamentous and granular layers. The mats were stored in plastic bags and refrigerated until

sectioning for microscopy. In this study, the top pigmented layers and a section of older material

from the unpigmented layers were studied for both mat morphotypes. Representative SEM

images of all four mat samples are shown in Figure 6.

imaging as well as one ~2 cm thick section of a brown filamentous layer starting from ~4 cm

deep in the mat. This layer was chosen because there were visible vertical tufts, and because it

occurs deep enough in the mat that we can observe an entirely different generation of mat

growth. This allows us to test hypotheses about microbial mat succession after storm or other

disturbance events as well as what from the active polygonal mat might be preserved in the

degraded polygonal mat that could enter the geologic record. From the flat mat, one ~2 cm thick

section was taken of the green and pink pigmented layers and one ~2 cm thick section of a tan

layer from ~4.5 cm deep in the mat was taken to again test hypotheses about preservation in the

geologic record. The unpigmented layers represent the old, buried mats that contain material that

might enter the geological record while the pigmented, active layers capture the processes that

all sample preparation and imaging took place. All four mat sections were fixed with 3.0%

formaldehyde and 1.5% glutaraldehyde in 0.1 M sodium cacodylate and 5 mM Ca

2+

, 5 mM

MgCl

2+

, 2.5% sucrose, pH 7.4, at room temperature for one hour. Samples were then washed

three times in 0.1 M cacodylate, 2.5% sucrose, pH 7.4, for fifteen minutes for each wash.

Afterwards, samples were post-fixed with Palade’s OsO

4

for one hour in the dark. 5 mL of

Palade’s 1% OsO

4

is composed of 1 mL Acetate-veronal sock, 1.25 ml 4% OsO

4

HCl, and 1.75 ml deionized water. After osmication the mat samples become discolored and it is

difficult to tell with certainty what was the original “up” direction. Therefore, 0.10 mm stainless

each of the active and old polygonal mat sections and each of the active and old flat mat sections,

twenty sites were sampled for imaging at intervals of roughly every 1-3 mm. Images were taken

from both the green and pink layers of the active mats. Two images were taken at each site, one

at ~1000x and another at around ~3500x. Images were taken at these two magnifications in order

to reduce bias towards either large or small organisms and features. High voltage mode was used

for all images. In all subsequent sections, discussion of the polygonal mat and flat mat refer to

the combination of results and analysis from both the green and pink layers of those mats.

2.3 Image analysis

All SEM images were analyzed using the image processing software JMicroVision. For

Different statistical methods were employed using Python for analyzing the point count

data and the filament width data. In order to analyze the point count data of the various features

and species/morphologies in the mats, two statistical methods were used to help visualize and

quantify the data. First, to visualize differences between the polygonal, flat, old polygonal, and

old flat mats, principal component analysis (PCA) was applied to the data in a variety of

different ways to investigate relationships in the data. Across the four different mat samples, nine

different microorganism morphologies and features were identified during point counting. The

relative abundances of these features were then calculated. The most abundant of these features

by far was amorphous organic matter, the classification used for the decayed and featureless

organic matter in the mats. In order to explore how similar the mats are based on both the whole

The second statistical method used to analyze the point count data and determine how

similar the different mat samples are to one another was non-metric multidimensional scaling.

The Bray-Curtis dissimilarity metric is commonly used in ecology to evaluate the dissimilarity

between two different sites. The Bray-Curtis dissimilarity metric was chosen for this study

because it can be used on relative abundance data from different locations, or in this case,

different microbial mat types and ages. The Bray-Curtis dissimilarity metric returns a percent

dissimilarity between two mat types and/or layers (e.g. percent similarity between polygonal and

flat mats, or percent similarity between the polygonal green layer and the polygonal pink layer)

analyzed by PCA and Bray-Curtis dissimilarity are in Table A1.

For the filament widths, the Welch’s t-test, or the unequal variances t-test, was used to

test the hypothesis that that the cyanobacteria filament widths in the polygonal mat are

indistinguishable from those in the flat mat. The Welch’s t-test was also used to determine if the

cyanobacteria filament widths are indistinguishable between the old mat samples and the active

mat samples in order to help determine if the old samples represent buried polygonal or flat mats.

The Welch’s t-test was used because of the differing variances and sample sizes of filament

widths from the different mat types and layers (Table A2). Because Welch’s t-test assumes

normality, the test was performed on filaments with widths from 0-5 μm, a range where the data

are approximately normal. However, Welch’s t-test is robust to non-normality (Ruxton, 2006),

sklearn.decomposition.PCA; Bray-Curtis dissimilarity: scipy.spatial.distance.braycurtis). See the

SciPy documentation for more information on those functions.

3. Results

3.1 Point counting

The most abundant mat morphology/feature identified during point counting across all

four mat samples was amorphous organic matter, which accounts for >83% of features observed

in the mats (Fig. 7 and 8). The polygonal mat and its green and pink layers have both the least

amorphous organic matter and the most cyanobacterial filaments. The old mats have the most